The ability to manipulate the genetic content of living cells via transformation is one of the core technologies of the field of biotechnology. By altering the genetic repertoire of a cell, one can augment its normal capabilities, cause the cessation of some normal cellular activity or cause cells to perform activities which are entirely outside of their normal range of action. These possibilities have been exploited in many ways. Currently, the simplest and most common use of genetic manipulation is the reprogramming of cells to synthesize large amounts of desirable protein products. This kind of manipulation is usually performed on either unicellular organisms or on cells which have been abstracted from multicellular organisms and then maintained in culture as single cell types. As the number of available genes and the extent of knowledge about regulation of genes has expanded, greater and greater interest in altering the genetic constitution of normal multicellular organisms have been evoked. However, one of the primary limits to the alteration of multicellular organisms has been the technology available to carry out transformation.
The primary means for delivery of DNA into living cells are: cellular uptake of DNA precipitates, micro-injection of DNA into single cells, electro-fusion, insertion of DNA into cells by micro-projectiles coated with DNA, and cellular uptake of DNA from the surrounding solution following exposure of the cells to a strong electric pulse (i.e., electroporation or electro-transformation).
Cellular uptake of DNA precipitates requires that special non-physiological conditions be attained in the fluids surrounding the cells for relatively long periods of time (hour/s). This is not practical when dealing with whole multicellular organisms, as they have very effective systems which condition the fluids surrounding their cells.
Micro-injection of DNA into single cells is inefficient, tedious and very limiting in the number of cells which can be treated, since each treated cell must be individually handled.
Electro-fusion is a means by which exogeneous genetic material is introduced into a host plant (See U.S. Pat. No. 4,832,814 to Root). This insertion is accomplished by either permeabilizing the cell membrane to allow entry of genetic material or fusing the host cell with a cell containing the desired genetic material. Electro-fusion has many limitations and has not been found to apply to all plant cells. (See U.S. Pat. No. 4,822,470 to Chang).
Insertion of DNA into cells using DNA-coated micro-projectiles (See U.S. Pat. No. 4,945,050 to Sanford and Wolf) is another technology that has been used to genetically engineer plants. In one procedure the cells to be treated are exposed to a high vacuum, followed by a blast of gas and detritus from an explosive event. A vacuum is required so that the accelerated micro-projectiles will retain sufficient velocity to pierce the target cells during their journey from the site of acceleration to the site of impact. The exposure to gas and debris from an explosive event arises from the need to use an explosion to achieve the high degree of acceleration required to give the micro-projectiles the requisite kinetic energy to pierce the target cells. Both of these conditions impose severe limitations to applying the projectile method to cells in whole organisms. This technology also requires that plants go through a tissue culture stage, and again the conditions for regenerating whole plants form plant cells are not well established for most plants. See D. E. McCabe, et. al., "Stable Transformation of Soybean (Glycine Max) by Particle Acceleration", BIO/TECHNOLOGY, Vol. 6, Aug. 1988, page 923; and J. C. Sanford, "The Biolistic Process", TIBTECH, Dec. 1988 (Vol. 6, page 299).
Cellular uptake of DNA following exposure of the cells to an electric pulse can take place in surroundings which are not too far from physiological, on time scales which are on the order of milliseconds. The current designs for devices to distribute the requisite electric pulses all utilize two electrodes whose surfaces are separated from each other by distances greater than 1 millimeter. H. Potter, "Electroporation in Biology: methods Applications, and Instrumentation", Analytical Biochemistry, 174, 361-373 (1988).
Furthermore, present day electroporation devices require that the target cells (i.e., the host cell and its transformant(s)) be placed into a cavity (e.g., a cuvette) formed between the electrodes. See U.S. Pat. No. 4,695,547 to Milliard et al; U.S. Pat. No. 4,764,473 to Matschke et al; U.S. Pat. No. 4,882,281 to Milliard et al; and the four U.S. Pat. Nos. 4,946,793; 4,906,576; 4,923,814; and 4,849,089 to Marshall. Such designs require that larger and larger voltages be applied to the plates with increasing cavity width to accomplish the same field strengths, and also place geometrical limits on what targets can be treated, by requiring that they be placed between electrodes. In fact, electro-transformation is routinely carried out on cells in suspension. If the cells were originally in a tissue, they are first dissociated to single cells or small aggregates of cells and then treated.
The conventional process and the equipment used in practicing electroporation also have practical drawbacks and shortcomings. Dangerous high voltages are required. Occasional arcing through bacterial suspensions can explode sample cuvettes and create bacterial aerosols. These kinds of technical problems require improvements in both power supply and sample cell design and performance. B. M. Chassy et al, "Transformation of Bacteria by Electroporation", TIBTECH, Dec. 1988 (Vol. 6, page 303).
In addition, attempts to insert new genes into cereal grains, such as corn, wheat and rice, have fallen short of total success. Researchers at the U.S. units of such companies as Ciba-Geigy Ltd. and Sandoz Corp., both Swiss owned, have inserted new genes into corn plants but the mature plants have been unable to reproduce. Smaller biotechnology companies like DNA Plant Technology Corp. In Cainnaminson, N.J., also are attempting to genetically alter corn. J. E. Bishop, "Scientists Report Inserting Gene into Corn Plants that Stay Fertile", Wall Street Journal, Technology & Science, Jan. 24, 1990.
One interesting application of electroporation has been the transformation of plant germplasm by introducing DNA into pollen and mating ova of a plant line with the transformed pollen (See U.S. patent application Ser. No. 350,356 filed on May 8, 1989 and assigned to U.S. Secretary of Agriculture). This procedure avoids the complications of regeneration from protoplasts or tissue culture. Still another means for breeding new plant varieties is to use microspores. Microspore culture is the technique of growing plants from immature pollen cells (See U.S. Pat. No. 4,840,906 ; to Hunter and European Patent 301,316 to E. Heberlebor et al).
Therefore, much remains to be done in the field of electroporation. Easier to use processes, inexpensive equipment and the ability to practice electroporation outside of the laboratory are improvements that would be welcomed by both the biotechnology industry and academic molecular biologists.